Regulation analysis by cis reactivity, RACR

ABSTRACT

Methods of detecting affinity interactions between at least two molecules of interest are provided. The method comprises: a. forming a plurality of interactors by coupling each molecule of interest with at least one nucleic acid moiety comprising an identification sequence element and at an association element; b. promoting an association between at least two nucleic acid moieties from different interactors to form a plurality of unique associated oligonucleotides, wherein each nucleic acid moiety may form more than one unique associated oligonucleotide, and wherein each unique associated oligonucleotide comprises at least two identification sequence elements derived from the at least two nucleic acid moieties; c. selecting the plurality of unique associated oligonucleotides; and d. subjecting the selected associated oligonucleotides to an analysis that permits detection of the at least two identification sequence elements. Similar methods directed to detecting functional interactions, libraries of interactors employable in the present methods, and kits comprising those libraries are also provided.

RELATED APPLICATION

This application claims priority to U.S. Provisional application Ser.No. 60/697,415, filed on Jul. 8, 2005.

FIELD OF THE INVENTION

The invention relates to methods for detecting affinity-basedinteractions and functional-based interactions, and the relativestrengths of those interactions between molecules of interest. Librariesof interactors are constructed wherein the interactors comprise at leastone molecule of interest coupled to at least one unique nucleic acidmoiety. The inventive methods allow detection, interactoridentification, and determination of relative strengths of allinteractions possible within a library of interactors. Kits enablingrapid and efficient application of the inventive methods are alsoprovided.

BACKGROUND OF THE INVENTION

To identify molecular networks, information concerning at least twomolecular species must be gathered since there are at least twomolecules involved in any interaction. Current high throughputtechniques generally resolve one of the interaction partners in a highthroughput fashion while the other interaction partner is limited tomuch fewer species. There is a need for methods which permit highthroughput screening and identification of all partners of aninteraction.

The human genome contains approximately 30,000 genes, disregardingsplice variants and post translational modifications this correspondsapproximately to 30,000 proteins and 4.5*10⁸ possible protein pairs. Ingeneral, if there are n molecules of interest in the library, then thereexists (n²+n)/2 possible interaction pairs in the network. Thecapability of investigating vast libraries and networks like these wouldbe greatly enhanced by the development of molecular interaction-basedmethods wherein high throughput information may be generated for bothpartners of the interaction.

To analyze complex libraries a readout platform which enables dataacquisition in high throughput fashion is required. Protein analysis viamicro-array techniques can be achieved by converting information aboutprotein interactions into nucleic acid-based information, which ishighly amenable to analysis with microarrays. Microarray analysis is aconventional platform which enables cost effective data generation in ahigh throughput manner. A method of protein interaction analysis whichis successfully adapted for microarray analysis is highly desirable. Thepresent inventors have developed such an adaptation wherein molecularinteractions are detected by detecting a combination of at least twonucleic acid tags, one from each molecule, wherein at least one suchcombination is required for signal output in one microarray feature.

Several other approaches exist in the art which can be utilized forinteraction screening, but which differ from the present inventivemethods in significant ways.

Protein microarrays use micro-scale, spatial localization foridentification of different molecules. The format allows detection ofmolecules in high throughput if target molecules in solution areinvestigated by probes organized on a solid phase. Targets can belabelled with a detectable function and the presence of the detectablefunction in one array feature reflects the presence of the target in thesample, assuming that no cross reactivity between probes are present.Examples of this approach are expression arrays and antibody arrays. Theassumption when using these arrays is that each target in solution onlydisplays affinity to one defined probe on the microarray. Wheninvestigating molecular interactions the affinities are not known perdefinition. To identify molecular interactions with regular microarrayanalysis one detectable function is required for each target insolution.

The number of detectable functions which can be resolved then becomeslimiting. Generally only one or two detectable functions are usedalthough there are approaches which potentially could resolve more theyare far away from the number of molecules which can be identified byposition in the array. This limits the capacity of informationextraction per experiment to the number of detectable functionsmultiplied by the library size, e.g. if a library has 500 members andthe approach can detect two detectable functions 250 arrays need to beperformed to extract all possible interaction information. Further on,the microarray platform suffers from the disadvantage that one partnerof the protein pair needs to be immobilized on a solid phase whichpotentially can disturb the interaction and/or the protein conformation.While protein microarrays permit analysis of one member of aninteraction in high throughput and can provide information aboutinteractions, the data extraction is cost and labour effective. Theanalysis is performed on a solid phase and interaction studies arelimited by the number of resolvable detectable functions which alsolimits the possibility of inter- or intra-library interaction screens.

Another known method is the yeast two hybrid system which utilizestranscription factors to investigate protein interactions. Manytranscription factors contain two distinct functional regions, one whichbinds a specific DNA sequence and one which recruits the transcriptionmachinery to activate a proximal gene. Yeast two hybrid systems utilizethese features to investigate protein affinity interactions. One proteinor protein library is fused with the DNA binding domain forming the“bait” and a second protein or protein library is joined to theactivating domain forming the “prey”. The fusions are constructed on theDNA level and the two constructs are then co-expressed in yeast. If twoproteins which have affinity for each other are expressed in the samecell they will activate transcription. Affinity interaction can then bedetected by reporter gene activation due to reconstitution of thetranscription factor. The two major approaches for yeast two hybridinteraction analysis is the “array approach” and the “exhaustivescreening” approach. In the array approach yeast clones expressingdifferent baits are ordered in an array so that the identity of theprotein in each clone can be deducted from position. The array is thenmated with yeast expressing one prey protein of interest. The matedclones are then cultured on selective media and protein interactions canbe identified without cloning and sequencing from the position ofpositive clones on the array. The exhaustive screening approach mate onebait clone with a library of prey proteins, the mated library is thencultured on selective media and positive clones are sequenced toidentify which prey proteins have interacted with the bait. The majordrawback of the exhaustive screening approach is the time and cost ofsequencing all the positive clones, which combined with the highintrinsic level of false positives increase the time and costdramatically, compared to the array approach. Hence, library-libraryscreens are generally not considered.

While the yeast two hybrid systems are advantageous in that they aregenetic systems and thus do not require protein synthesis, and they canperform high throughput of one of the interaction partners, sequencingof positive clones is necessary for high throughput library screeningapproaches, which, combined with an intrinsic high frequency of falsepositives becomes problematic.

Display techniques such as phage display, ribosome display, RNA display,SELEX, cis display and covalent display are used to isolate affinitybinders from libraries. These techniques are based on a large library ofpotential interaction partners (binders) which are allowed to interactwith one partner (target). The target is typically immobilized on asolid phase and then allowed to interact with the library of binders.Members of the library which do not bind to the target are discarded andbound members are regenerated. Then the procedure is iterated until onlyone or a few library members remain. These are then analysed foraffinity towards the target. The different display techniques primarilydiffer in what molecule type is used to create the library and how thebinders are regenerated. Display techniques can be used to identifyaffinity interactions but these are generally limited to a low number oftarget molecules. Some display techniques like phage display have thepotential to identify interactions between libraries but the analysis ofthe interactions are done by cloning and sequencing which is very timeconsuming and labour intensive. A majority of the display techniqueslike e.g phage display are genetic systems and do not easily adapt toother molecules than proteins.

To enable selection of binders based on libraries other than peptide,protein or nucleic acid, libraries, DNA templated synthesis (DTS)approaches has been developed. In this technique, nucleic acids can beattached to low molecular compounds in order to establish identificationof the compound after selection of a library for a desired property. Iftwo compounds are attached to nucleic acids which are complementary,hybridization of these nucleic acids will force the two compounds inproximity and elevate the relative local concentration of the compounds.Thus, the two compounds will be more prone to react than if they werenot associated. This approach has also been utilized for multi stepreactions, where libraries of compounds joined to nucleic acid tags aredirected to serially react with a compound attached to a templatenucleic acid. The synthesized library can then be screened with respectto a desired property and selected compounds can then be identified.Further on, the identity of compounds synthesized by DTS can beidentified by microarray hybridization of the nucleic acid templatewhich directed the synthesis and to which the product is also attached.

In DTS, two molecules are forced together by using nucleic acids toallow the molecules to interact to a higher degree than if they were notbrought in proximity. For example, in the DTS-based method taught byKanan et. al (Kanan et. al Nature 2004), one of the molecules forcedtogether is attached to a “template” nucleic acid which specificallydirects the second compound by direct hybridization of the nucleic acidattached to the second molecule. Thus the “template” nucleic acidcontains both tags used to identify the compounds which are forcedtogether. To create interactions between all members of a library with nmembers with this approach, the number of “template” oligonucleotideswhich have to be synthesized are in the range of n²/2, since onespecific oligonucleotide has to be synthesized for each individualinteraction on the library.

However, the present invention provides a method whereby the nucleicacid encoding the tags to identify both members of the interaction areformed upon joining of the two molecules. Hence, to investigate allinteractions in a library of n members the number of oligonucleotidesrequired is in the order of n. The combination of molecules in DTS isnot combinatorial in the sense that all library members potentially mayinteract. It becomes combinatorial due to the presence of all specificpairs DNA templates which in turn direct the interactions. If thespecificity of these interactions fails, the link between the compoundphenotype and the nucleic acid genotype also fails. Thus, in the DTSapproach, the link between nucleic acid identification tag and themolecules in the interaction depend on correct hybridization of one tagmolecule to the template nucleic acid. If any cross hybridization occursthen the link between the tag and the identity of the interaction isunreliable.

In the current invention the nucleic acid formed by the NAM associationencodes the combination of identification sequences. Therefore the linkbetween the identification sequences in the associated nucleic acidmolecule and the phenotype of the molecule is more effectivelymaintained.

The utilization of combinatorial association of different nucleic acidtags instead of pre-synthesis of all different combinations alsoprovides further advantages. The amplification of the nucleic acid canbe performed so that only nucleic acids which have been joined to createa pair of molecules are amplified by, e.g., introducing one primer siteon each arm. Nucleic acids which remain unassociated will thus not beamplified. In the DTS approach all template nucleic acids can serve asPCR templates even though they have no interaction partner.

A method referred to as “proximity ligation” has been recently described(International Patent Application Ser. No. WO0161037, U.S. PatentApplication Ser. No. US2002051986). According to this method, targetsare detected by utilizing two or more binders, e.g. antibodies, withknown affinity to the target. The method is based upon theco-localization of the binder pair in the presence of a target. Thistarget brings the binders into proximity, enabling ligation of nucleicacids located on the binder pair. Thus the joining of the nucleic acidbecomes elevated in the presence of the target molecule. The nucleicacid can subsequently be quantified and the amount of nucleic acidcorresponds to the amount of target.

The primary embodiment of WO0161037 and US2002051986 aims at detectionof a defined target. Thus all the affinities in the system are known andthere is always at least one molecule, the target, which does not have anucleic acid attached to it. Moreover, the invention utilizes two ormore binders which bind their unlabelled analyte pair-wise in apredefined manner. Several pairs might be used in the same reaction butthey are always analyzed in a target specific pair wise fashion, not ina combinatorial fashion.

The current inventive method differs from proximity ligation in that itdoes not detect or quantify a predefined target in a sample. The presentinventive methods do not utilize molecules with predefined affinitiesbut rather the reverse; the inter-molecular affinities are retrieved asa result of the inventive method.

In the current invention the association of the nucleic acid iscombinatorial and the novel nucleic acid produced by this association isthen identified to yield information concerning the molecularinteractions in the library. This enables interrogation of all intra- orinter-library member interactions. The proximity ligation methods alwaysinclude at least one molecule which is not attached to a nucleic acidlabel and this “target” is the molecule which is detected. Thus theco-localization/proximity of the binders and thereby the nucleic acid inthe assay arises from the presence of a target molecule.

Finally the information gained from the inventions differssignificantly. In the present inventive methods, combinations ofinteracting molecules may be detected and quantified. The proximityligation art, on the other hand, teaches high throughput screening ofinhibitors for a defined binding event. For example, a protein affinityinteraction is identified first by some other method and proximityligation is employed to find an inhibitor for the known affinityinteraction. The proximity ligation screening approach involvesattaching nucleic acids to the two proteins which participate in thepredefined interaction. Then different potential inhibitors are added tothe reaction. These potential inhibitors are not labelled with nucleicacids and their action is monitored by observation of a reduced signalfrom the two labelled proteins. The pre-defined affinity reagents arelabelled with nucleic acids and a pre-defined pair of nucleic acids isanalyzed.

There is a need in the art for methods capable of investigating affinityand/or functional interactions within libraries of molecules whereinsuch interactions are not previously established to exist, and methodsfor quantification of any detected interactions.

SUMMARY OF THE INVENTION

Accordingly, the present invention is directed to methods fordiscovering, as well as detecting molecular interaction networks such asaffinity interactions or functional interactions, e.g.,enzymes-substrate pairs. Significantly, the invention allowsinvestigation of all possible interactions within or between librariesof molecules, such as proteins, and also permits relative quantificationof the detected interactions. Information about the interactions isderived through the use of nucleic acid tags which facilitates highthroughput analysis, preferably by microarray analysis, however otheranalysis platforms may also be used. Typically, each feature on themicroarray corresponds to a molecular interaction between two specificmolecules in the library.

Each molecule of interest (MOI) in the library is tagged with a nucleicacid tag, referred to herein as a nucleic acid moiety (NAM). If twomolecules interact the combination of their nucleic acid tags is thenselectively enhanced. The oligonucleotide formed from the combination oftwo NAMs is referred to herein as an associated oligonucleotide.

One embodiment is directed to a method of detecting an affinityinteraction between at least two molecules of interest. The methodcomprises: (a) forming a plurality of interactors by coupling eachmolecule of interest with at least one nucleic acid moiety comprising anidentification sequence element and at an association element; (b)promoting an association between at least two nucleic acid moieties toform a plurality of unique associated oligonucleotides, wherein eachnucleic acid moiety may form more than one unique associatedoligonucleotide, and wherein each unique associated oligonucleotidecomprises at least two identification sequence elements derived from theat least two nucleic acid moieties; (c) selecting the plurality ofassociated oligonucleotides; and (d) subjecting the selected associatedoligonucleotides to an analysis that permits detection of the at leasttwo identification sequence elements.

According to this embodiment, an “interactor” library is developed. Eachinteractor consists of the molecule of interest (MOI) tagged with atleast one unique nucleic acid moiety (NAM). The NAM comprises, interalia, an identification sequence making downstream detection possible.It is the associated oligonucleotides which are subjected to qualitativeand quantitative analysis, as a single associated oligonucleotidecorresponds to an interaction between the two MOIs tagged with the NAMsthat comprise that associated oligonucleotide. The library ofinteractors (NAM-tagged MOIs) is first allowed to interact andequilibrate. As the MOIs associate based on any affinity(s) betweenthem, the corresponding NAMs associate in a proximity dependent mannerincidental thereto.

Another embodiment of the present invention is directed to a method ofdetecting functional interactions between at least two molecules ofinterest. The method comprises: (a) forming a plurality of interactorsby coupling each molecule of interest with a nucleic acid moiety, thenucleic acid moiety comprising an identification sequence element and anassociation element, wherein an affinity exists between the nucleic acidmoieties; (b) forming a plurality of cis-reactive cells wherein acis-reactive cell comprises at least two interactors bound in proximityto one another by an associated oligonucleotide formed from the affinitybetween at least two nucleic acid moieties, wherein the associatedoligonucleotide comprises at least two identification elements derivedfrom the at least two nucleic acid moieties; (c) subjecting theplurality of cis-reactive cells to conditions which stimulate a desiredfunctional interaction having a detectable trace; (d) selecting allcis-reactive cells exhibiting the detectable trace; and (e) subjectingthe associated oligonucleotides from the cis-reactive cells selected in(d) to an analysis that permits detection of the at least twoidentification sequence elements.

As in the affinity embodiment, each MOI is tagged with a unique NAM toform an interactor. However, in some functionality-based embodiments,the NAMs are designed to so that a pre-existing affinity exists betweenthem. MOIs are independently tagged with the NAMs. Again, the interactorlibrary is permitted to interact and equilibrate, creating a secondarylibrary of “cis reactive cells” (CRCs) wherein each CRC consists of MOImolecules brought into proximity by the affinity between theirrespective NAMs, and co-localized by the association of their respectiveNAMs. Subsequently the function to be investigated is activated. Whenactivated, MOIs will primarily act upon the other members in the CRC dueto the proximity created by the physical linkage. In some embodiments itmay be desirable to dilute the concentration of CRCs in order to lessenthe probability of trans activation of the function. That is, activationbetween, rather than within, CRCs.

Following functional activation the secondary library is filtered forCRCs which contain MOIs that have been altered by the activation.Selection of positive (that is, altered) CRCs can be done by, e.g.,affinity purification of the library of CRCs with respect to amodification introduced by an enzymatic reaction. The CRCs are thenidentified by the sequence motifs in the NAMs, thus both members of afunctional interaction can be identified, for example, pairs of novelenzymes and novel substrates.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

Schematic description of one embodiment for affinity interactiondetection. A) Library morphology; a library with 1, 2, . . . , n MOIs,each associated with a defined NAM with nucleic acid tags 1, 2, . . . ,n. B). The library is allowed to interact by affinity to induceco-localization of the MOIs and the respective NAMs. C) The associationfunction of the NAMs is activated and NAMs brought into proximity byaffinity between their respective MOIs are associated to for a novelassociated oligonucleotide comprising nucleic acid from both of theoriginating NAMs. D) Selective amplification of the different NAMcombinations. NAMs which are not associated will not yield anyamplification product for subsequent detection.

FIG. 2

Schematic description of one embodiment for functional interactiondetection. A) Library morphology; a library with 1, 2, . . . , n speciesof MOI, each MOI species is associated with a defined NAM with nucleicacid tags 1, 2, . . . , n. B) Construction of CRCs. The libraryassociation function is designed to co-localize the library members in acombinatorial fashion to create all possible CRC variants between the nlibrary members ((n−n²)/2 unique combinations). C) The functionalinteraction to be detected is activated and reaction conditions arecontrolled to restrict the functional interactions to substantiallywithin the CRCs. D) Selection. Selection is carried out with respect toat least one detectable alteration introduced by the functionalinteraction. E) Amplification of the selected CRCs and subsequentdetection and analysis of the nucleic acids.

FIG. 3

Schematic description of the library design in the exemplary approachdescribed for affinity interaction. A) The NAMs are produced from a seedPCR product and unique NAMs are synthesized with tailed PCR primerscomprising one of a plurality of nucleic acid tags. An associationfunction is located in one primer and a conjugation function is locatedin the other. B) library morphology after conjugation to MOI. Theassociation element in this case comprises a protruding end which allowsselective blunt end ligation.

FIG. 4

Schematic description of the library design in the exemplary approachdescribed for detecting a functional interaction. A) NAMs beforeconjugation to the MOI are depicted, including two separatesub-populations of NAMs comprising potential kinases and substratesrespectively, one with the forward primer and one with the reverseprimer. Each NAM comprises a PCR primer, identification element and anassociation element. B) Library morphology after association of theinteractors to construct CRCs is illustrated. Each CRC is joined byaddition of a polymerase that stabilizes the hybridization of therespective 3′ ends and incorporates the nucleic acid tags and PCRprimers into one associated oligonucleotide by polymerization.

FIG. 5

Non-limiting examples of association of NAMs. Arrows indicate nucleicacid 3′ ends. FIGS. 5( a-d) illustrate ligation approaches. Thestructure in 5(e) can be associated with either ligation orpolymerization with a polymerase comprising 5′ exonuclease activity.5(f) can be associated with polymerization. 5(g) illustrates associationby introduction of a functional group which reacts to create a nativenucleic acid. In 5(h) reactive functions are used which associate thenucleic acids by a link separate from nucleic acid.

FIG. 6

Examples of two tag microarray detection. Black sequences represent ageneral sequence common to all targets and red sequences representidentification tag sequences. 6(a) illustrates hybridization of a targetwhere the two tag elements are separated by a general sequence that isnot complementary to the microarray probe. 6(b) illustrateshybridization of a target comprising two identification elements notseparated by any spacing or general sequence. In 6(c) the identificationtags are located in the 3′ and 5′ ends with the hybridized targetforming a circular structure that could be ligation-templated by themicroarray probe. In 6(d) the target is hybridized to form a circularstructure similar to 6(c) but with a general spacing sequence at theends of the target and in the microarray probe, an detection can bemediated by sandwich hybridization of a third nucleic acid or by directlabelling of the target. FIG. 6( e) represents detection of a targetwith two joined tags by ligation of a third nucleic acid comprising adetectable function. In 6(f) the detection is mediated bypolymerization, for example, by a exonuclease deficient polymerase,rather than ligation. 6(g) represents detection of the structure formedin 6(c) by ligation of the circular structure, polymerization andsubsequent detection by addition of a third labelled nucleic acid. Thepolymerization event could also be detected by labelled nucleotides.

FIG. 7

This represents results from the example for reaction discovery. Threetag microarrays from three experiments with different reactionconditions are displayed. A higher intensity represents detection ofhigher concentrations of the tag combination. Each hetero-interaction isrepresented by two separate microarray features.

FIG. 8

Illustration of a single stranded associated oligonucleotide or singlestranded amplification product thereof. Two separate single strandedoligonucleotides comprising a type IIS restriction enzyme recognitionsites are hybridized to the general sequence flanking the identificationelements. The restriction enzyme cleavage occur between theidentification element and the general sequence. FIG. 8( b) Illustratesthe single stranded DNA produced by the restriction enzyme cleavage withidentification elements in the 3′ and 5′ end.

FIG. 9

Diagrammatic illustration of microarray features representing 3′ arm and5′ arm tag combinations resulting from an interaction context. Data isplotted on a grid including the different 3′ and 5′ NAMs with the signalto noise (feature signal/sum of all feature signals) plotted on thez-axis. The arms with thiol groups are 3′A and 3′B and 5′C and 5′D.

DETAILED DESCRIPTION OF THE INVENTION Terms & Definitions

As used herein, the term “combinatorial association” refers toassociation of a plurality of molecules to create a plurality ofcombinations of those molecules.

As used herein, the term nucleic acid moiety (NAM) refers to a nucleicacid molecule which is coupled to at least one molecule of interest(MOI). A NAM comprises at least one unique identification nucleic acidsequence so that detection of that sequence infers involvement of thecoupled MOI. A NAM also comprises at least one element which enablesassociation with other NAMs. A NAM may be single stranded DNA, doublestranded DNA, single stranded RNA, double stranded RNA or combinationsthereof. The NAM may contain modified nucleotides or nucleotideanalogues. The NAM may be derived from synthetic, biochemical,biological synthesis or combinations thereof.

As used herein, the term “molecule of interest” (MOI) refers to amolecule about which interactive information is desired. An MOI iscoupled with at least one NAM. In one embodiment each MOI is coupledwith a single unique NAM. Examples of suitable MOIs include, but are notlimited to, whole proteins, protein domains, peptides, peptoids,functional enzymes, low molecular weight compounds, small molecules,polymers, non-proteinaceous cellular products, virus particles, cellsmetabolites, lipids, carbohydrates, nucleic acids, inorganic compoundsor combinations thereof. In one specific embodiment the MOI comprisesproteins. In any library of MOIs, not all the MOIs may solely comprisenucleic acids. In a more specific embodiment, the MOIs may comprisefactors regulating transcription, non-limiting examples includeenhancers, repressors, and isolators) and DNA sequences. A library ofsuch MOIs may be investigated for affinity interactions. In veryspecific embodiments the DNA sequences are derived from genomicsequences or comprise random elements. The DNA sequence MOIs maycomprise genes involved in transcriptional regulation and the presentinventive methods may be employed to reveal interactions between DNA andproteins to enable, for example, deduction of how the transcriptionalunits regulate transcription by binding to DNA.

As used herein, the term “small molecule” refers to a non-peptidic,non-oligomeric organic compound either synthesized in the laboratory orfound in nature. Small molecules, as used herein, can refer to compoundsthat are similar to complex natural products which nature has selectedthrough evolution, however, the term “small molecule” is not limited tothese compounds. Rather, a small molecule is typically characterized inthat it contains several carbon-carbon bonds, and has a molecular weightof less than 1500, although this characterization is not intended to belimiting for the purposes of the present invention. Examples of “smallmolecules” that occur in nature include, but are not limited to, taxol,dynemicin, and rapamycin. Examples of “small molecules” that aresynthesized in the laboratory include, but are not limited to, compoundsdescribed in Tan et al., (“Stereoselective Synthesis of over Two MillionCompounds Having Structural Features Both Reminiscent of NaturalProducts and Compatible with Miniaturized Cell-Based Assays” J. Am.Chem. Soc. 1998, 120, 8565) and United States Patent Application20030082830 “Synthesis of Combinatorial Libraries of CompoundsReminiscent of Natural Products”, the entire contents of which areincorporated herein by reference.

As used herein, the term “interactor” refers to at least one MOI coupledwith at least one NAM.

As used herein, the term “Cis Reactive Cell” (CRC) refers to anassociation of at least two interactor moieties, joined by an associatedoligonucleotide. In a specific embodiment a CRC comprises twointeractors.

As used herein, the term “interactor library” or “library ofinteractors” refers to a plurality of interactors.

As used herein, the term “Cis reactive cell library” or “library of cisreactive cells,” or “secondary library” refers to a plurality of CRCs.

As used herein, the term “association of NAMs” refers to an associationbetween the NAM components of interactors which results in a moleculecontaining nucleic acid sequences originating from the associated NAMmolecules. This combination of NAMs is referred to herein as an“associated oligonucleotide” and, in specific embodiments, comprises theidentification sequences for two component NAMs. The association may becovalent or non-covalent and occurs between at least two NAMs. Inspecific embodiments, the association occurs pairwise between two NAMs.

The types of interactions which may be detected with the currentinvention include, but are not limited to, interactions based onaffinity, enzymatic activity including kinase, glycosylation,phosphatase and ubiqutinylation activity, fusion, covalent bondformation, structural alteration, degradation, and/or subunit addition.A person of ordinary skill in the art will recognize that an“affinity-based” interaction may include any interaction which resultsin non-random assortment of molecules due to any basis other thanrepulsion.

The current invention aims to retrieve information about molecularinteractions. Interactions can be of any type between molecules such ase.g. affinity, enzymatic, structural, degrading, fusion, addition of one(or more) groups. The foundation of the interaction analysis is theincreased probability of a reaction between molecules if they arebrought in close proximity by molecular co-localization. Theco-localized molecules will be prone to interact in cis rather theninteract with separate molecules in trans with the presumption that therelative concentration of the co-localized molecules is higher then theconcentration of other molecules in the solution. In the case ofdetection of affinity interactions the co-localization is introduced bythe affinity interaction between the MOIs which then enhances theprobability of the respective co-localized nucleic acids to associate.Functional interactions are detected by bringing the MOIs into proximitywith one another through an independent assorting means. For example,MOIs may be brought into proximity with one another incidental toaffinities between the NAMs, and then co-localized by association of theNAMs to form interactors. This increases the probability that allpossible MOI groupings occur and are exposed to the functional trigger.A detectable function is then triggered.

The feature of intra-molecular enhancement of reaction kineticsfrequently appears naturally in biological systems like, for example,transcription activation where affinities co-localize the transcriptionmachinery to a gene initialized by binding of a transcription activator.The present inventive methods adapt such an approach in vitro as amolecular technique for analysis of molecular interactions.

According to the present inventive methods, interaction information isessentially encoded into nucleic acids which then are detected bynucleic acid analysis techniques. This is achieved by tagging each MOIwith a NAM comprising a unique nucleic acid identification sequence. (Insome embodiments more than one MOI may be tagged with the same NAMspecies). The downstream association of NAMs form a noveloligonucleotide molecule encoding the interaction. This approach allowscombinatorial analysis of all potential interactions in a library if alldifferent tag combinations can occur.

The following detailed description of the Interactor Library is dividedinto the following sections for clarity: components; construction;interaction analysis; filtering; and readout.

Interactor Library

Library Components

The current invention is based upon the construction of a library ofmolecules referred to herein as interactors. These library memberscomprise at least two distinct parts which are coupled, one part is themolecule of interest (MOI) and one part is the nucleic acid moiety(NAM).

Molecule of Interest (MOI)

The MOI component comprises molecules including, but not limited to,whole proteins, protein domains, functional enzymes, low molecularweight compounds, polymers, non proteinaceous cellular products, virusparticles, cells metabolites, lipids, carbohydrates, nucleic acids orcombinations thereof. The library may consist of a heterogeneouspopulation of MOI types. Minimally, the MOI component must meet tworequirements, it must be coupled with at least one uniquely identifiableNAM, and not all MOIs in the library can be nucleic acids.

Nucleic Acid Moiety (NAM)

The NAM may comprise single stranded DNA (ssDNA), double stranded DNA(dsDNA) single stranded RNA, double stranded RNA or combinationsthereof. It may include any type of modified nucleotides or nucleotideanalogues or other types of molecules which can be incorporated intonucleic acids. The coupling between a NAM and an MOI to form oneinteractor may be non covalent but covalent association is desirable.Each NAM is coupled to at least one MOI and it may associate with otherNAMs in the library.

Interactors

One interactor in the library comprises at least one MOI coupled to atleast one identifiable NAM. The inventive methods enable retrieval ofinformation concerning interactions between these interactors in vitro.The retrieved data can then be translated into information about theinteractions of the MOIs.

Library Construction/Coupling of MOI and NAM

The inventive methods prepare and utilize libraries of interactors asdescribed above. These libraries can be synthesized in several differentways. The construction of the interactor library includes coupling of atleast one MOI to at least one NAM. Generally the coupling means may bespecific or unspecific with respect to coupling position and couplingsmay be performed in trans and cis. In other embodiments an interactorcomprises more than one MOI and at least one NAM.

Coupling of MOIs and NAMs in Trans

It is possible to construct an interactor library merely by mixing aplurality of MOIs with a plurality of NAMs and permitting them toincubate and couple under conditions that favor coupling. Possiblemechanisms of coupling include, but are not limited to, chemical,enzymatic or affinity based mechanisms. Preferably the coupling betweenthe MOI and the NAM is covalent and stable or at least of high affinity.The coupling of the MOI to the NAM may also include addition of one orseveral types of bi-functional linkers. The coupling may be specific ornonspecific.

Nonspecific coupling includes means wherein the exact position of theNAM might not be controlled or known and wherein several NAMs mightcouple to one MOI. However, unspecific coupling of the NAM to the MOImight interfere with or alter the interaction phenotype of the MOI.Therefore specific coupling may be desirable when possible. Specificcoupling includes methods where the position of the NAM on the MOI iscontrolled and/or known. To control localization of the NAM a reactivefunctionality may be introduced on the MOI during or after synthesis ofthe molecule, whether synthesis is chemical, enzymatical, in vitro or invivo. This will secure NAM coupling with the MOI at defined position(s)on the MOI. Depending on the nature of the MOI, specific coupling of theNAM may be performed downstream of MOI synthesis without modification ofthe MOI if there is a novel site(s) on the MOI to which the coupling canbe directed.

Examples of Specific Coupling Means

Generally, specific coupling may be accomplished by introduction orutilization of a site(s) on the MOI to which a specific coupling can bedirected. The site can be used for direct coupling of the NAM orcoupling to a third molecule which then can be coupled with the NAM. Thenature of the association can be of covalent or non covalent as long asthe link between the MOI and the NAM is maintained. Differentinteractors in the library may be produced with different approached forassociation of the respective MOI and NAM molecules depending on thenature of the interactor.

One example of a specific coupling means is the generation of a specificsite by intein splicing. Several examples of this which may be utilizedin the present inventive methods are disclosed in Takeda et al. BioorgMed Chem Lett 14, 2407-2410 (2004), Southworth, et al. Biotechniques 27,110-114, 116, 118-120 (1999), and Burbulis et al. “Using Protein-DNAChimeras to Detect and count small numbers of molecules” 2, 31-37(2005), all of which are incorporated fully herein by this reference.Generally, this approach creates a site for specific chemicalconjugation in either the N-terminal or the C-terminal of a protein. Anintein approach which could be used for both MOI purification andconjugation has been commercialized by New England Biolabs under thetrademark IMPACT-TWIN. Specific coupling of the NAM to the MOI may alsobe performed by introduction of a fusion partner to the MOI which hasaffinity or enzymatic activity towards the NAM or vice versa. A specificexample of this is the introduction of the hAGT protein as a fusionpartner to the MOI and addition of the hAGT substrate to the NAM. hAGTthen covalently binds the substrate on the NAM. This has been used toconjugate different molecules to proteins fused to hAGT as disclosed inKeppler et al. Methods, 32, 437-444 (2004), also incorporated fullyherein by this reference.

A specific site may be introduced on the MOI to which a function forcoupling then can be introduced downstream of synthesis. An example ofthis approach is the utilization of biotin ligase as taught in Duffy etal. Analytical Biochemistry 262, 122-128 (1998), the disclosure of whichis fully incorporated herein by this reference. Briefly, a signalpeptide is incorporated into the MOI during synthesis, which then isrecognized by the biotin ligase. The biotin ligase recognizes thepeptide and adds a biotin to the MOI. This biotin can then be used forcoupling of the NAM by introducing an affinity molecule such asstreptavidine or avidine on the NAM. The introduction of the biotin maybe performed in vitro or in vivo.

Examples of Unspecific Coupling Means

Unspecific coupling includes the use of chemical coupling to sites onthe MOI which yield a coupling between the NAM and the MOI where theexact position of each NAM is uncertain and where the coupling may occurto several different sites on the MOI. These approaches would typicallyinclude one reagent (A) which can couple to the NAM and one reagent (B)which can couple to the MOI and a subsequent step where A and B areassociated. There is also the possibility to associate A and B first orutilize a molecule which binds both the NAM and the MOI. A person ofordinary skill in the art will appreciate that the diversity of thesemolecules is quite high and the choice will depend upon the nature ofthe MOI. Specific nonlimiting examples of functional groups which can beused for this type of coupling include NHS esther, malemide, Azide,Alkyne, amine, or thiol. For more examples see; Double-AgentsCross-Linking Guide (#1600918) from Pierce. Coupling can also bemediated by a reactive group on the NAM which reacts directly with theMOI or vice versa.

Coupling Between MOIs and NAMs in Cis

Instead of individual construction of the library interactors, thecoupling between the NAM and the MOI can be performed during thesynthesis of the MOI. The synthesis is controlled so that it willproduce coupling with at least one defined NAM. The interactor synthesiscan be performed in multiplex, i.e. several interactors can besynthesized in the same reaction. This could potentially speed up thelibrary construction significantly.

Nonlimiting examples of this include the approach used in CIS display orCovalent display as disclosed by Reiersen et al. Nucleic Acids Res 33,e10 (2005), or Odegrip, R. et al PNAS 101, 2806-2810 (2004), thedisclosures of which are fully incorporated herein by this reference. Alibrary of proteins is produced and during production the proteinproduct is specifically associated to its own DNA by a protein fusionpartner. Variants of this approach have been published whereby thecoupling is covalent or non covalent. In a modified approach this couldbe used to create libraries where the protein serves as the MOI and thethereto coupled DNA molecule serves as a NAM.

Another approach is the use of the mRNA display technique disclosed inWilson et al. PNAS 98, 3750-3755 (2001), fully incorporated herein bythis reference. According to this approach a pool of proteins isproduced from a pool of mRNA templates and the protein products arecoupled with the mRNA they were produced from by a pyromycinmodification at the end of the mRNA. The protein then serves as the MOIand the coupled mRNA as the NAM. A modified variant of this exists wherethe protein product is linked to a cDNA instead of the mRNA.

Another approach which creates a physical link between the RNA and theprotein is ribosome display, taught by Hanes & Pluckthun in PNAS 94,4937-4942 (1997), the disclosure of which is fully incorporated hereinby this reference. However in this particular coupling technique,coupling is mediated by the ribosome which is a large complex with thepotential to interfere with the assay. The design of the coupled nucleicacid could be adapted to the current invention in these systems and thenucleic acid component may be modified post synthesis by, e.g.,restriction endonucleases. In the genetic protein based systems thenucleic acid sequence encoding the proteins could be utilized as thenucleic acid identifying sequence for downstream detection. Constantsequences present on all nucleic acids in the library could be utilizedfor amplification.

A person of ordinary skill in the art will appreciate that any methodwhich utilizes a function which couples the DNA or RNA with the proteinproduct during synthesis could fulfil the purpose of creating librariesof interactors in multiplex employable herein. This has the additionaladvantage of ensuring integrity between the genotype and the resultantphenotype in display techniques. The important feature in theseapproaches is that the coupling between the nucleic acid and the MOIoccurs in cis. Thus, the binding of the nucleic acid primarily occurs tothe protein product synthesized from that nucleic acid. For couplingbetween DNA and protein this is mediated by the fact that the DNA isattached to the protein product by the transcription and translationmachinery.

The desired MOI-NAM coupling may be achieved by a variety of meansincluding fusion of MOIs to nucleic acid binding proteins which bindback to the nucleic acid upon synthesis and addition of molecules to theRNA or DNA which bind the synthesized protein or an attached fusionpartner. The binding may be directly or indirectly mediated by abifunctional linker, that is, an intermediate molecule holding the DNAand protein together. This intermediate molecule may range in size froma small molecule as defined herein, to a very large ribosome. The ciscoupling of a nucleic acid to a desired protein could also be mediatedby in vivo expression of, for example, plasmids, phages or viruses. Ifthe nucleic acid encoding the protein product is utilized as the NAM,constant motifs can be used for amplification purposes and MOI encodingmotifs for identification. One specific embodiment of the presentinvention is directed to methods of detecting affinity interactionsbetween at least two molecules of interest, wherein at least onemolecule of interest comprise a protein that is an expression product ofa nucleic acid molecule, and wherein at least some of the plurality ofinteractors is formed by coupling the protein with a nucleic acid moietycomprising an identification sequence element and an association elementderived from the nucleic acid molecule. This provides a library ofinteractors wherein each interactor comprises a molecule of interestcomprising a protein that is an expression product of a nucleic acidmolecule, coupled with a nucleic acid moiety tag, the tag comprising anassociation element and an identification sequence element, wherein theidentification sequence element and the association element derived fromthe nucleic acid molecule.

DTS, described in Kanan et. al Nature 431, 545-9, herein incorporatedfully by this reference, permits multiplex synthesis of MOIs other thanproteins. DTS may be utilized to synthesize libraries encoded by nucleicacids and these library members can subsequently be utilized asinteractors in the current invention.

Interaction Analysis

The present invention provides methods which permit analysis of theinteractor library with respect to interactions between the interactormembers, and, therefore, by inference, between the MOIs. One embodimentprovides analysis based primarily on affinity interactions between theMOIs. Another embodiment provides analysis based primarily onfunctionally-based interaction between MOIs. Affinity interactions areinteractions where two or more members of the library display a bindingaffinity towards each other. Functional interactions are interactionswhere two or more members affect each other in some detectable way andmay include affinity interactions, though a person of ordinary skill inthe art will appreciate that there are functional interactions whichdisplay very low or transient affinity.

Affinity Interactions

One embodiment of the present invention is directed to a method ofdetecting an affinity interaction between at least two molecules ofinterest. The method comprises: (a) forming a plurality of interactorsby coupling each molecule of interest with at least one nucleic acidmoiety comprising an identification sequence element and at anassociation element; (b) promoting an association between at least twonucleic acid moieties from different interactors to form a plurality ofunique associated oligonucleotides, wherein each nucleic acid moiety mayform more than one unique associated oligonucleotide, and wherein eachunique associated oligonucleotide comprises at least two identificationsequence elements derived from the at least two nucleic acid moieties;(c) selecting the plurality of associated oligonucleotides; and (d)subjecting the selected associated oligonucleotides to an analysis thatpermits detection of the at least two identification sequence elements.

Broadly, affinity interactions are defined as interactions between twoor more molecules that induce a non-random distribution of thesemolecules in a solution comprising these molecules. Thus, the moleculeswhich display the affinity will more frequently be close to each otherthen if their distribution were random. It is noted in particular that anon-random distribution per se may be due to a repulsion between theMOIs such that the MOIs are more frequently separated than in a randomdistribution.

Affinity interactions within the interactor library are analyzed byallowing the library members to mix and equilibrate under conditionswhich permit affinity interactions. Following this step association ofNAMs is actively promoted. The association of NAMs is performed so thatassociation between two NAMs depends on proximity of the NAMs, with theproximity being due to an affinity interaction between their respectiveMOIs. Thus MOI affinity interaction induced proximity or co-localizationof NAMs induces a higher association frequency of those NAMs compared toa control where the MOI affinity is absent. Downstream of theassociation, the associated NAMs (the associated oligonucleotide) canoptionally be amplified and subsequently detected, by, for example,microarray analysis.

A person of ordinary skill in the art will recognize that a strongeraffinity between two MOIs generally induces more frequent association ofthe NAMs. This enables a relative quantification of the interactionaffinity strength between interactors and, by inference, the MOIs.

Functional Interactions

Another embodiment of the present invention is directed to a method ofdetecting functional interactions between at least two molecules ofinterest. The method comprises: (a) forming a plurality of interactorsby coupling each molecule of interest with a nucleic acid moiety, thenucleic acid moiety comprising an identification sequence element and anassociation element, wherein an affinity exists between the nucleic acidmoieties; (b) forming a plurality of cis-reactive cells wherein acis-reactive cell comprises at least two interactors bound in proximityto one another by an associated oligonucleotide formed from the affinitybetween at least two nucleic acid moieties, wherein the associatedoligonucleotide comprises at least two identification elements derivedfrom the at least two nucleic acid moieties; (c) subjecting theplurality of cis-reactive cells to conditions which stimulate a desiredfunctional interaction having a detectable trace; (d) selecting allcis-reactive cells exhibiting the detectable trace; and (e) subjectingthe associated oligonucleotides from the cis-reactive cells selected in(d) to an analysis that permits detection of the at least twoidentification sequence elements.

Functional interactions are defined as interactions between moleculeswhere the nature of at least one of the partners which participate inthe interaction deviates from the nature of that partner when the otherparticipant(s) of the functional interaction is/are present inconditions permitting the functional interaction. Functionalinteractions may include an affinity interaction.

Functional interactions include interactions with functions such as,e.g., kinase, phosphatase, glycosylation, deglycosylation,ubiqutinylation, deubiqutinylation or other paired activities where onemember adds or removes a molecule(s) from the other member. Additionalnonlimiting examples include interactions with altering activities suchas cleavage, fusion, and inducement of a structural alteration in one ormore members of the interaction. Significantly and advantageously, thepresent invention permits identification of both partners of afunctional interaction. The criterion for detection of functionalinteractions by this approach is that the functional interaction iscapable of being detected or selectively enhanced to permit detection.

The approach to identify functional interactions is similar to that foridentification of affinity interactions but differs in some keyfeatures. When employing the previously discussed embodiment directed toaffinity interactions, the association function of the NAMs is activatedafter interactors are permitted to display any affinity interactionswhich may detectably exist. In the case of detecting functionalinteractions the MOI association activity is controlled and theinteractors in the library are forced together by association of theirrespective NAMs, NOT primarily between any affinities between the MOIs.One way to achieve this is by introducing an affinity between thedifferent NAMs in the library by hybridization. This will createco-localizations of two or more interactors through the association ofthe NAMs. The result of the forced NAM association is a secondarylibrary of cis reactive cells (CRCs). A CRC is a co-localization of twoor more interactors which are associated by their NAMs (associatedoligonucleotide). Thus the MOI components of the same CRC are brought inproximity although the MOIs might not have any intrinsic affinity.However, it is contemplated that in some embodiments the CRCs may becreated by relying on intrinsic MOI affinities or a combination of bothMOI affinity and NAM affinity.

If two individual MOIs are members of a functional interaction pair andthe conditions favour this function, the MOIs in one CRC, being incloser proximity to one another, will interact more frequently with oneanother than with MOIs in other CRCs. It is important that theinteractor concentration in the library be maintained lower than therelative concentration between the MOIs in the CRC.

Once the CRC library is constructed the functional interaction beinginvestigated is stimulated. This can be accomplished by, for example,addition of a substrate or a shift in reaction conditions. The MOIs inthe CRC will primarily act on each other in cis since they arephysically linked, but they could potentially act in trans with MOIs inother CRCs. Hence, measures are taken to ensure that any functionalinteractions remain substantially restricted within the CRCs. Suchmeasures may include, but are not limited to: temporal limitation of thefunctional activity; competition with the reverse functionality; anddilution of the associated library.

In one specific embodiment, temporal limitation of the functionalinteraction is achieved by adding a component essential for theactivation in a milieu where this compound is rapidly degraded. Thedegradation of the component may be chemical or enzymatic. In a furtherspecific embodiment, competitive limitation is achieved by activatingthe functional interaction in a milieu where it is reversed. The actionwhich reverses the functional interaction could be of chemical orenzymatic nature. If the functional interaction includes addition of amolecular group or alteration of a structure the reaction conditions canbe maintained so that the added group hydrolyses or the structurereverts to the initial state quickly. Employing enzymes which remove thegroup added by the functional interaction may also be an option. If afunctional interaction is present within a CRC it might be counteractedby the reaction conditions or by the addition of compounds but theintrinsic functional activity of the CRC will immediately counteractthis. On the other hand, if a member of a CRC acts in trans on aseparate CRC this functional interaction will be reversed by thereaction conditions and the probability of a repeated functionalinteraction in trans is low. Thus, an equilibrium that favour functionalinteractions within CRCs can be achieved.

In another specific embodiment, the CRC library is diluted subsequent toassociation of the interactors but prior to activation of the functionbeing investigated. Interactor association will be more efficient athigher concentrations, but this also elevates the risk of transinteractions between the CRCs once the function is triggered. Dilutingthe concentration after formation of the CRCs will lower theconcentrations of CRCs in the library while the proximity between MOIswithin the CRCs remains.

The library is then analyzed with respect to which CRCs display thedesired functional interactions. The functional interaction beinginvestigated is limited to those functions that are detectable.

Association of NAM Molecules

Association of interactors is a key feature of the current invention inboth the embodiment for detection of affinity interactions and theembodiment for detection of functional interactions. The binding of theinteractors by their respective NAM association elements forms novelmolecules, herein referred to as associated oligonucleotides, comprisedof the nucleic acid tags comprising the identification sequences derivedfrom the component interactors. Analysis of the associatedoligonucleotide molecule formed from the combination of these tagspermits identification of the interactor MOI components.

It is desirable that the NAM association results in a moleculecomprising nucleic acid derived from each of the associated interactors.There are several approaches to achieve this and the examples describedbelow and in FIG. 5 serve to illustrate some approaches, though thisshould not be construed as excluding other approaches. Association byenzymatic ligation may be achieved as illustrated in FIGS. 5( a-e).Ligation substrates may include single stranded nucleic acid templatedby a third nucleic acid, double stranded nucleic acid with protrudingends templated by a third nucleic acid, ligation of single strandednucleic acids, templated ligation where a separate nucleic acid isintroduced into the ligation product or ligation of double strandednucleic acid with complementary protruding ends. Exemplary associationapproaches for polymerization include substrates comprising doublestranded nucleic acid with complementary protruding 3′ ends, or singlestranded nucleic acid with complementary 3′ ends, as illustrated inFIGS. 5( d) and 5(f).

Association may also be accomplished via chemical rather than enzymaticligation. Chemical ligation may be performed by providing nucleic acidswith reactive groups on the ends which allow association by chemicalreaction. The chemical reaction may result in a nucleic acid of nativecomposition which can serve as, for example, a polymerization substrate,or a nucleic acid derivative which does not serve as polymerizationsubstrate. Two illustrative approaches for chemical association areillustrated in FIGS. 5( g) and (h). Other non-limiting examples of NAMassociation approaches which could be used are gap fill polymerizationand subsequent ligation and gap ligation as described in Lizardi et al.Nat Genet 19, 225, 232 (1998), or invader cleavage and subsequentligation as described in Lyamichev et al, Science 260, 778-783 (1993),both of which are fully incorporated herein by reference. In a specificembodiment the nucleic acid is DNA but other nucleic acids may also beused.

The association approach utilized depends on the qualitative assaydesign. An illustrative example is the introduction of PCRamplification. If PCR amplification of associated NAMs is desired theassociation may preferentially combine NAMs comprising the forwardprimer with NAMs comprising the reverse primer to form amplificationsubstrates. Selective association may be achieved by introduction of theforward primer to a portion of the interactor library with theassociation element in the 3′ end and the reverse primer in anotherportion of the interactor library with the association element in the 5′end. This would be desirable since both primer motifs are required foramplification of associated NAMs by PCR.

In one embodiment, selective association approaches which excludehomo-association permit construction of interactor libraries withinteractors comprised of more then one NAM per MOI. If homo-associationis not excluded, then the homo-associations of NAMs associated to thesame MOI be very efficient and potentially render the library useless.

In one embodiment, two sub type libraries are constructed wherein eachsubtype comprises a different NAM and the NAMs of one subtype canassociate only with NAMs from the other subtype. If the associatedlibrary is enhanced with PCR subsequent to association each NAM sub typecomprises one of the PCR primer motifs. In the case of affinityinteractions this design also allows association of several NAMs to eachMOI, since unique NAMs are unable to associate. For functionalinteractions, the design allow presentation of all possible interactorcombination in one mixing step, such as when interactor librariescomprising different NAM subtypes are pooled. If the NAM association isefficient and/or of high affinity this has the potential to overrideaffinities present between MOIs since each interactor will associatewith any interactor comprising a compatible NAM and this may occurbefore equilibrium is achieved with respect to the individual MOIaffinities. Subsequent to construction of CRCs, the interactor librarycan be diluted to reduce the effects of MOI affinities between CRCs.

Association of Nucleic Acids in for Affinity Interaction Analysis

For investigation of affinity interactions it is desirable to use NAMscomprising ssDNA or dsDNA. For investigation of affinity interactions itis important that the different NAMs do not display too high of anintrinsic affinity toward one another other. The affinity between thedifferent association elements of the examples illustrated in FIGS. 5(a) to (f) depend on the length of the complementary sequences of theassociation elements, and the addition of a third nucleic acid whereapplicable, except in 5(c) where no complementary sequence is present.Low affinity may by achieved by restricting each complementary sequenceto 1-10 nucleotides. Association of NAMs by addition of a thirdtemplating nucleic acid can advantageously be performed by templateaddition in high concentration (e.g. >100 fold) compared to theinteractor concentration. This will provide template mediatedassociation of NAMs proximal at the moment of addition, while individualNAMs will hybridize to templates individually. Once all NAMs havehybridized to a template nucleic acid, this hybridization can blockfurther template mediated dimerization of NAMs by intrinsic stability.This approach may allow utilization of template nucleic acids withhigher stability.

Association of Nucleic Acids for Functional Interaction Analysis

CRCs are constructed by the association of NAMs. The association mayadvantageously force different MOIs together and therefore affinitybetween the different NAMs is desired. In the exemplary illustrations5(a, b, d, and f) this is achieved by introducing stable complementarysequences in the association elements.

When a third templating nucleic acid is employed for construction ofCRCs, this may advantageously be added in equimolar or slightly higher(e.g. <5 fold excess) concentrations compared to the total interactorconcentration. This may introduce an equilibrium where a majority of theNAMs are associated.

According to another embodiment, association between NAMs with lowintrinsic NAM affinity is employed to construct a CRC library whereinthe frequency of each CRC depends on the relative affinities of theinteractors. This permits investigation of both affinity interactionsand functional interactions with the same library. The CRC library mayalso be enriched for CRCs subsequent to association to avoidtrans-interactions from individual interactors.

The association reaction can also be regulated by reaction conditions.For example, in an exemplary library comprised of proteins theprotein-protein affinity interactions may be destabilized byintroduction of high salt concentrations, which also stabilizes thenucleic acid interactions. Thus the inter-interactor affinities areshifted to depend on the nucleic acids.

Filtering and Selection

Affinity Interactions

The selection in the affinity embodiment of the inventive methodsprimarily concerns elimination of interactors which are not associatedwith any other interactors and maintaining the associated interactors.This can be achieved in several ways. One way is to perform selectiveamplification of associated NAMs, for example, with PCR, wherein thereis one primer on each NAM so that only joined NAMs will produceamplification products. This approach might necessitate two types ofNAMs per MOI (one with each primer motif) to enable identification ofall interactions in the library including homo interactions.

A second amplification-based selection method is to cut the respectivearms with a restriction enzyme and circularize the nucleic acid with ageneral template. Upon a second ligation, ligated arms will form acircular structure while individual arms will form a linear structure.The circular nucleic acid can then selectively be amplified by rollingcircle amplification, a well-known technique in the art. Another way isto immobilize one part of the library to a solid phase and then performthe association of the NAMs. After the association, washing may removeall interactors which have not associated with any immobilizedinteractors. All molecules may or may not be present in solution orimmobilised on the solid phase. The interactors may be immobilized in aspatially organized way or in a random fashion. Following washing of thesolid phase the combinations of NAMs may be analyzed with or withoutamplification. One example of a suitable procedure would be elution ofthe interactors and amplification of the associated NAMs.

According to another embodiment, associated NAMs are selected bydegrading any NAMs which are not associated. An example of this approachwould be addition of an exonuclease which degrades any free nucleic acidends. If the NAMs have been associated with a ligase the associated NAMswill not have any free 5′ or 3′ ends and not be amenable to degradation.

Functional Interactions

To detect functional interactions in a pool of CRCs the CRCs whichdisplay functional interactions must be selected for detection and/oranalysis. It will be appreciated by one of ordinary skill in the artthat this step will be modified according to the nature of thefunctional interaction that is being investigated. As used herein, a“filter” or a “filtering process” is the means employed to selectivelyseparate the desired CRCs. Nonlimiting examples of filters forfunctional interactions include: affinity purification of the form whichhas undergone alteration in the functional interaction; detection of theCRCs by labelling with affinity reagents which detect interactors whichare included in the functional interaction; and introduction of amodified molecule which becomes incorporated by the functionalinteraction where the modification permits selective detection.Downstream of the filtering process of CRCs the compositions ofassociated NAMs are detected and/or quantified.

One example of a suitable filtering process is, affinity purification.Filtering of positive CRCs in the case of an interaction which includesaddition of a group by one member of the interaction to the other membersuch as a kinase or a glycosylation interaction between enzyme andsubstrate may be accomplished by affinity purification of CRCs withrespect to the added group. In the specific example of a kinaseinteraction the library of CRCs can be affinity purified with aphosphate specific antibody. The specificity can be controlled to aphosphorylated amino acid, peptide motif or a whole protein depending onthe assay. CRCs which do not contain the modification will be discardedand the CRCs with a modification will be retained and detected. Each CRCwith a modification can be assumed to contain two members with theactivity screened for, unless a homo interaction occurs. So in the caseof kinase activity each selected CRC contains one kinase and onesubstrate thereof. The affinity filtering could also serve as a negativeselector where the undesired molecules remain on the solid phase and thedesired ones are collected. As non-limiting examples, this type ofselection would be suitable in the case of phosphatase activity,glycosylation/deglycosylation, ubiquitinylation/deubiquitinylation andfor any other interaction which serves to add or remove molecules orparts from one of the interaction partners as long as there are aspecific affinity reagent for the molecule or part which is added orremoved.

If the functional interaction results in a structural change in one ofthe partners to the interaction, affinity purification can be performedwhich only recognizes the altered portion. If no affinity reagents areavailable for the molecule which is added or removed an affinityfunction could be attached to the group which is added. For example, aprotein like ubiquitin may be fused with one interaction partner and theadded ubiquitin may be labelled with biotin which may then be affinitypurified with streptavidine. However the “filtering” is not necessarilythrough affinity purification and can also be, for example, aconditional detection of the NAMs. If the detection output is coupled tothe result of the interaction, for example, with an affinity reagent forthe modification, only the CRCs with positive interaction pairs will bedetected.

Another example of a suitable filtering process is conditionaldetection. The CRCs can be hybridized to a DNA oligonucleotide tag arraywhere all possible combinations of identification sequences in thelibrary are present. The different combinations of associated NAMs arespatially separated by the hybridization. To identify which NAMcombinations represent a CRC with a positive interaction pair the arraywith the CRCs can be stained with a labelled affinity reagent specificfor the interaction. However this approach requires that the MOIs arepresent during detection and thus no amplification of the associatedNAMs is possible. A detectable function may also be associated with amodification, more specifically, for example, a fluorescent orradioactive label can be introduced by the modification, which morespecifically comprises, in kinase interactions for example, aradioactive phosphate group.

Detection

In one embodiment, detection or readout of the library interactions isachieved by analysis of the NAM combinations, both qualitatively andquantitatively, by nucleic acid analysis. In a more specific embodiment,the nucleic acid composition is analyzed using microarrays. Otherembodiments contemplate other known and unknown methods of nucleic acidanalysis or include detection of molecules attached to the NAMs or therespective MOIs. The important feature is that at least two bits ofinformation are retrieved, each representing one partner of theinteraction.

Nucleic Acid Based Readout

In one specific example, the analysis of interactions present in thelibrary is accomplished by analysis of the combinations of the NAMelements by microarray analysis. The analysis of the associatedoligonucleotides is similar both for the affinity interaction assay andfunctional interaction assay, once the CRCs of the functionalinteraction assay are filtered/selected for those CRCs which containfunctionally positive interactors as described above. In someembodiments, the detection of the associated oligonucleotides ispreceded by an optional amplification step.

Amplification Methods

To increase the sensitivity and speed up the analysis, the associatedoligonucleotides can be amplified by conventional nucleic acidamplification techniques such as, for example, polymerase chain reaction(PCR), rolling circle amplification (RCA), strand displacementamplification (SDA), nucleic acid sequence based amplification (NASBA),RNA transcription or invader assay. If the NAMs comprise RNA, reversetranscription may be used to translate these sequences into DNA. Otherembodiments contemplate additional methods, known and unknown, ofnucleic acid amplification. In the case of affinity interactions theamplification can also be used as a step to select for associated NAMs.

When PCR is utilized for amplification of associated NAMs, two primermotifs are required. In one specific example, PCR is used foramplification of associated NAMs wherein one primer motif is located oneach NAM so that associated NAMs are selectively amplified. However PCRamplification requires two types of NAMs per MOI, one for eachrespective primer, to enable amplification of all intra-library MOIcombinations. If inter-library interactions are detected, the librariescan be designed to comprise separate primer motifs.

RCA amplification of associated NAMs requires synthesis of a circularpolymerization substrate. Conversion of the associated oligonucleotideconstructed upon NAM association to a circular substrate can be achievedby removing the MOIs by, for example, restriction enzyme digestion andallowing circularization of the associated oligonucleotide by templatedintramolecular ligation.

Sequence Information Retrieval Approaches

The pertinent information which must be retrieved from the associatedoligonucleotides is the identity of the two identity sequence motifswhich provide information about the identity of the MOIs correspondingto that associated oligonucleotide. Thus at least two pieces ofinformation need to be decoded. There are several ways to do this andmost of them are either sequencing based or hybridization basedapproaches.

Hybridization Based Approaches:

The use of microarrays to identify nucleic acid sequences in complexpools is well-known and has been adapted to high throughput platforms,for example, in expression profiling. The utilization of oligonucleotidearrays has increased the specificity and the introduction of in situoligonucleotide synthesis has enabled very high throughput analysis bycheap probe synthesis of very complex microarrays. Currently the majorplatforms for microarray hybridization to oligonucleotides arefabricated by either robotic deposition of chemically synthesizedoligonucleotides or in situ synthesis of the oligonucleotides on thearray. Separate platforms for microarray readout also exist, such as theIllumina platform, wherein oligonucleotides are attached to beads whichsubsequently organize on a solid support by self assembly.

The present inventive methods detect different tag combinations. If alibrary consists of n different interactors, each with a differentidentification sequence in the NAM, there would be (n+n²)/2 potentialcombinations including homo-interactions, and (n²−n)/2 potentialcombinations without homo-interactions. If two subtypes of NAMs areutilized for one MOI library, each subtype with a unique set ofidentification sequences, the number of tag combinations will be n²,where each interaction is represented by two separate unique tagcombinations except the homo-interactions.

Microarrays with probes for all these nucleic acid tag combinationscould be manufactured for readout. The number of probes needed increaseexponentially with the number of library members according to theformula above. However in situ synthesis of microarrays can produce verycomplex arrays which could be used for any interaction study since thenucleic acid tag set generally is independent of the MOIs.

The general approach to detecting the NAM combinations by microarraybased readout involves construction of a microarray wherein all possibletag combinations in the library are present. The associatedoligonucleotides, or amplification products thereof, are allowed tohybridize to the array. Preferably the melting temperature of theidentity sequence elements should be designed so that the differencebetween hybridization of one element and two elements is maximized.Other criteria that increase hybridization stringency could also beintroduced to maximize specificity, such as, for example, enzymaticdiscrimination steps. Non-limiting examples of these include ligationand/or polymerization.

The design of the microarray-based dual tag detection includeshybridization of a nucleic acid comprising at least two separate tag oridentification sequence motifs to array probes with their respectivecomplements. Illustrative examples of microarray hybridization designsare provided in FIG. 6. Hybridization of the two identification sequenceelements t_(x) and t_(y) (red in FIG. 6) may be accomplished byhybridization with or without a general sequence (black in FIG. 6)present on the targets. The elements may be hybridized as a continuoussequence (6 b) or with a spacing sequence between the elements (FIG. 6a). The elements may also be designed to form a circular structure uponhybridization (FIGS. 6 c and d). The specificity of the detection can beenhanced by introduction of enzymatic steps. 6 e represents ligation ofa general detection oligonucleotide. Incorporation of nucleotidescomprising a detectable function is illustrated in 6 f and solid phasepolymerization and subsequent detection by hybridization of a generaldetection oligo is illustrated in FIG. 6( g).

Specificity

The specificity of the detection of combinations of identifying elementsmight have to be enhanced if the stringency from the hybridization isinsufficient. The hybridization of identification element combinationsdiffers from regular hybridization of single elements. If there are n*2NAMs (two NAMs per protein) and n² array identification elementoligonucleotides on the array the different array elements can bereferred to as t_(x,y) where x is the identity of one identificationelement position and y the identity of the other identification element.Thus x and y can be any element between 1 and n. The combination of thearbitrary identifying elements a and b will thus complementary to thearray element t_(a,b) but they will also be partially complementary tothe array elements t_(x,b), and t_(a,y).

The specificity of the hybridization should substantially ensure thatthe combination a,b primarily hybridizes to the array element t_(a,b).Cross hybridization might occur to some or all of the array elementst_(x,b), and t_(a,y) to some extent, however. In a complex pool of tagcombinations which may have a high dynamic range of the differentcombinations the discrimination between two species may becomedifficult. If, for example, the combination a,b is present in 10,000fold excess over the combination a,c the nucleic acid from the a,bcombination might cross hybridize to the array element t_(a,c) which isdedicated for the a,c combination. This specificity problem may besolved by, for example, the introduction of enzymatic discriminationsteps.

Enzymatic steps have facilitated specific detection of individualnucleotide sequences in the human genome by, for example, utilization ofa ligase (see Landegreen et al. Science 241, 1077-1080 (1988), Hardenbolet al. Nat Biotechnol (2003), Yeakley et al. Nat Biotechnol 20, 353-358(2002), fully incorporated herein by reference), or a polymerase (seeFan et al. Genome Research 14, 878-885 (2004), fully incorporated hereinby reference). This specificity should be sufficient for a syntheticsystem where the sequences of the nucleic acid sequences can be designedin silico.

Polymerase Based Discrimination;

To increase the selectivity with a polymerase the hybridized nucleicacids can be allowed to serve as primer or template for a polymeraseextension reaction including labelled nucleotides. If this is performedwith the proper polymerase which lack, for example, 3′ exonucleaseactivity, and the detection of the hybridized molecules depend on thisstep, the selectivity of detection is enhanced. Thus only nucleic acidswhich have hybridized to the array and can form a structure which canserve as a substrate for polymerization will be detected (FIG. 6 f).This approach has been utilized for microarray based discrimination ofsingle nucleotide variations in human genomic DNA (see Gunderson, K Let. al Nat Genet. 2005 May; 37(5):549-54, fully incorporated herein byreference).

Ligation Based Discrimination:

To increase the selectivity of detection by ligation the hybridizednucleic acid can be allowed to act as a template or substrate forligation. The detectable function can be added on a thirdoligonucleotide which can be joined to the surface complex by ligation.Only if the hybridization event yields a structure which together withthe third oligonucleotide, forms a substrate for ligation, willdetection be mediated. The ligation may be performed at the 5′ end, the3′ end, or both ends of the hybridizing nucleic acid (FIG. 6 e).

Ligation and Polymerization Based Specificity Enhancement.

Ligation can also be used in conjunction with polymerization to permitspecific detection. If the identifying elements are hybridized so that acircular structure is formed where the 5′ and 3′ end are injuxtaposition to each other, these two ends can be joined by a ligase toform a circular nucleic acid template in the array. This circularnucleic acid can then serve as a template for polymerization from thearray nucleic acid 3′ end. If a processive nucleic acid polymerase isused, for example, phi 29 polymerase, a concatemeric polymerizationproduct of several copies of the nucleic acid circle can be produced(see Hatch, A et al Genet Anal. 1999 April; 15(2):35-40, fullyincorporated herein by reference). These copies can then be detected byhybridization of an oligonucleotide attached to a detectable functionwhich hybridizes to the sequence between the identification sequences.This approach only yields signal output if a ligation substrate isformed (FIG. 6 g). Other non-limiting examples of ligation approacheswhich could be used in combination with polymerization are gap fill andsubsequent ligation as described in Lizardi et al. Nat Genet 19, 225,232 (1998), or invader cleavage and subsequent ligation as described inLyamichev et al, Science 260, 778-783 (1993).

Sequencing Based Approaches

Sequencing based approaches retrieve the nucleotide sequence of the twoidentifying elements. These approaches yield a very high complexity ofinformation and thus relatively few nucleotides need to be sequenced toenable identification of a unique element. For example the complexity offive nucleotides is 4⁵=1024 different unique sequences. Thus ifsequencing approaches are used very short identifying elements can beutilized. The length of the element used for identification can beadjusted to suit the complexity of the library being investigated.Identification of two different tags in a library of 1000 members and10⁶ combinations would require sequencing of at least 10 nucleotides perinteraction, and five nucleotides per MOI.

However, when sequencing complex pools of nucleic acids, measures haveto be taken to isolate the individual molecules or cloned productsthereof. This can be done with conventional techniques like molecularcloning into bacteria and subsequent Sanger sequencing. The sequencingof molecular identification sequences of approximately 10 nucleotidescan be speeded up significantly by using approaches similar to serialanalysis of gene expression (SAGE) where short sequences are joinedtogether before sequencing and several identifying elements can besequenced in one sequence read. Sequencing of complex pools can also beperformed by high throughput approaches like the 454 platform(www.454.com). This approach includes emulsion based clonal PCRamplification and subsequent sequencing on a solid phase. Approacheslike this permit very high throughput analysis of the readout.

Depending on the possible readout length these approaches may becombined with SAGE like polymerization of paired identificationelements. Solid phase sequencing of the joined tag motifs or possiblyconcatemeric products thereof would permit information retrieval of theinteraction frequency in digital form. However, the sequences combinedby the interaction have to be significantly more salient than thebackground frequency, otherwise the majority if the sequences retrievedwill only be random combinations. Sequence based detection of aninteraction which is 1000 fold less frequent than another will requiresequencing of 1000 identical sequences of the frequent one per copy ofthe rare one.

Interactor Based Detection

In some situations nucleic acid based amplification of the NAM might notbe desired. The different combinations of interactors may then beanalyzed directly. One advantage of this is that other identificationmoieties than the actual nucleic acid could be used or the MOIs may beidentified directly. Nonetheless, the readout approach requiresidentification of at least two separate pieces of information from theassociated interactors.

One way of retrieving information from two different species isutilization of a nucleic acid tag array for hybridization of thecombinations. In this approach the array would only contain individualtags on the array. Thus if there are n identifiers in the library thearray would consist of n spatially separated oligonucleotides.Subsequent to the NAM association and filtering, which could includeselection for combined NAMs, the complex of associated interactors ishybridized to the array. The localization of the interactor complexeswill reveal the identity of one of the members and the second member isidentified by a separate tag moiety, which may be of another nature thannucleic acid. For example, if fluorescent readout is chosen the secondtag could be decoded with sequential combinatorial hybridization asdescribed in Gunderson et al., Genome Research 14, 870-877 (2004)disclosed fully herein by this reference.

Another approach could be to identify the second tag by matrix assistedlaser desorption/ionisation time of flight mass spectrometry (MALDI TOFMS). According to this method, the identification elements in the NAMswould include molecular “tags” which could be identified with thisapproach. These tags would preferentially be non nucleic acid moleculesassociated with the NAMs. Depending on the nature of the MOIs thesemolecules could be identified by MALDI TOF MS directly without theutilization of a separate molecule for the detection.

EXAMPLES

The following examples are intended to illustrate specific embodimentsof the present invention and should not be construed as limiting theinvention as defined by the claims. All detection schemes encompassedwithin the scope of the present invention include combinatorialassociation of NAMs.

Example 1 Affinity Interactions

This example illustrates embodiments directed to detecting affinityinteractions.

The individual proteins to be investigated are produced by recombinantexpression and purified. These proteins serve as MOIs in the library andare coupled with NAMs to form interactors.

The NAMs are double stranded DNA molecules and comprise a primer motif,a nucleic acid identification sequence and an association sequence. Toenable amplification of all intra-library combinations, there are twosub populations of proteins created, one with the forward PCR primer andone with the reverse primer in their respective NAMs (FIG. 3). Thenucleic acid identification elements in the two sub-populationscomprised by the same protein are not identical, thus each protein is“encoded” by two nucleic acid identification elements in the library.

The individual NAMs are synthesized from two generic PCR products whichcomprise the general primer motif and spacer sequence. The nucleic acididentification sequence is introduced by performing a second PCR on thegeneric PCR product with where one primer contains a conjugationfunction and the other a 5′ tail encoding the nucleic acididentification element and the association element.

The coupling function is performed by a sticky end which is created byrestriction enzyme digest. The protruding end is created by utilizing arestriction endonuclease which produces a non-palindromic protrudingend. The NAMs encoding the forward primer are designed to have onepolarity of the protruding end and the NAMs encoding the reverse primerare designed to have the complementary protruding end. The designpermits association of several identical NAMs to one MOI withouthomo-ligation between the NAMs. The individual NAM subtypes are gelpurified and associated with the MOI by the conjugation element in oneof the primers. The proteins are recombinantly expressed with the IMPACTsystem from New England Biolabs and the conjugation function of the NAMPCR product is a Cysteine residue. C-terminal fusion of the protein inthe IMPACT system generates a SO₃ ⁻ group at the N-terminal of theprotein which then can be specifically conjugated to the Cysteine groupon the NAM. However IMPACT may perform poorly on certain members andadditional approaches for conjugation may also be utilized to includeall desired proteins.

Subsequent to interactor synthesis by individual coupling of the PCRproduct derived NAMs to the proteins, the individual interactors arepooled in 1×PBS with inclusion of bovine serum albumin and polyadenine.The interactors are allowed to affinity interact and are subsequentlyligated by addition of a ligase mix containing ATP, T4 DNA ligase TRISHCl pH 7.5 and MgCl₂. Following ligation the library is heated todeactivate the ligase and an aliquot of the library is transferred to aPCR reaction. PCR is performed with one biotinylated primer and one Cy3labelled primer. Subsequent to PCR amplification the fluorescent strandis purified by immobilization of the PCR product on dynabeads andelution of the fluorescent strand. The elution is mixed withhybridization buffer and co-hybridized to a microarray with a controlsample. The control sample is treated with protease degradation beforeligation and the fluorescent primer is Cy5 labelled. After hybridizationthe microarray is scanned and analysed with respect to the fluorescentratios.

The microarray comprises features with all combinations between tags onreverse primer and forward primer NAMs. Thus if 100 proteins areanalysed the number of features is 10,000, representing every libraryinteraction in duplicate.

Example 2 Functional Interactions

This example illustrates construction of a library of interactors andCRCs and detection of a specific enzyme-substrate functional interactionwhere one MOI acts as an enzyme and a second MOI is the substrate.

Enzyme substrate interactions can be difficult to distinguish withconventional approaches since the enzyme might have low or transientaffinity to the substrate. A specific choice of enzyme-substrate pair iskinases and their respective substrates. For screening proteins forkinase and kinase substrate properties a library of interactors isconstructed where the MOIs of the library are potential kinases andsubstrates respectively.

The interactor library comprises two sub-populations, the firstcomprising potential kinase MOIs and the second comprising potentialsubstrate MOIs. Each sub-population of a potential kinase is conjugatedto the 5′ end of a oligonucleotide subset comprising the forward primermotif for downstream amplification, the association element in the 3′end of the oligonucleotide and one of a plurality of nucleic acididentification elements. Each sub-population of a potential substrate isconjugated to the 5′ end of an oligonucleotide comprising the downstreamreverse primer motif, an association element in the 3′ end of theoligonucleotide and one of a plurality of nucleic acid identificationsequences (FIG. 4).

The NAM association function is a motif for polymerase assistedassociation at the ends of the oligonucleotides which are not conjugatedto the protein. The 3′ sequences are designed to be complementary andbrings the oligonucleotides comprising the forward primer and thereverse primer together. Upon polymerisation association is stabilisedand the respective tags and PCR primers are encoded into the samenucleic acid molecule. The kinases and substrates are proteins and theconjugation between these proteins and their respective oligonucleotidesare mediated by expression of the proteins by the IMPACT system andconjugation of oligonucleotides with a Cysteine residue. However properinteractor synthesis may require different expression and conjugationapproaches depending on the nature of the protein.

Following interactor synthesis CRCs are stabilized by the addition of apolymerase, nucleotides and the appropriate reaction conditions. 20 ntof the oligonucleotide 3′ ends of the two interactor sub populations arecomplementary and subsequent to hybridization the oligonucleotides serveas both polymerisation templates and substrates respectively. Thepolymerisation encodes the two identification sequences joined by thehybridisation into one nucleic acid molecule and also stabilise thehybridization of the oligonucleotides further. Polymerisation isachieved by addition of klenow fragment exonuclease deficientpolymerase, nucleotides and the appropriate reaction buffer.

The interactors are associated through polymerization to form CRCs andthe library is diluted so that the concentration of CRCs becomessignificantly lower than the relative concentrations between theinteractors in the CRC. To regulate the kinase activity apyrase is addedto the library before activation of kinase activity by ATP addition. Theappropriate relative concentrations of apyrase and ATP might have to betitrated depending on the kinase activity screened for. The apyrase willdegrade the ATP added to the CRC library and thus provide a temporallimitation of kinase activity. Subsequent to activation, the library ofCRCs is affinity purified with an antibody specific for a phosphorylatedamino acid or amino acid sequence. The library can be split into subsetswhich are investigated with separate affinity reagents. The selectedlibrary is then eluted and an aliquot is amplified by PCR comprising oneCy3 labelled primer and one biotinylated primer. The PCR product isimmobilized on streptavidine beads and the fluorescent strand is elutedand co-hybridized to a microarray with a control PCR Cy5 labelledproduct derived from a library which has not been affinity purified. Themicroarray contains features which are complementary to all kinase andsubstrate tag combinations in the library, representing all kinase andsubstrate interactions between the two libraries.

Example 3 Reaction Discovery

In one specific embodiment screening for reactivity between molecules isprovided. A library of molecules is subsequently screened for formationof chemical bonds between the library molecules. The procedure issimilar to discovery of affinity interactions, however, in thisembodiment, the interaction results in a covalent bond between themolecules. The embodiment enables reactivity screening in differentreaction conditions and/or in the presence of catalysts.

The molecules of interest are each tagged with a single stranded DNA NAMto form the interactor library. Two sub types of interactors areconstructed, comprising the same molecules of interest, one with freeNAM oligonucleotide 3′ ends and one with free NAM oligonucleotide 5′ends. These are constructed so that any free 3′ end can be joined withany free 5′ end by the addition of a DNA oligonucleotide template,complementary to each of the oligonucleotide sub types, by the additionof a ligase and under the appropriate reaction conditions. Each subtypealso comprises one PCR primer motif. Oligonucleotides with free 5′ endscomprise the forward primer and oligonucleotides with free 3′ endscomprise the reverse primer. All DNA NAM oligonucleotides also compriseone unique sequence element for identification.

Ten different molecules of interest are investigated for reactivity inthree different reaction conditions, thus there are 10*10=100 differentpossible reactions, each interrogated in three different reactionconditions.

The 10 interactors comprising NAMs with identical association elementsare pooled to form two populations, one comprising 10 interactors withfree 3′ ends one and comprising 10 interactors with free 5′ ends. Thesepools are subsequently mixed. The interactors are incubated inconcentrations of 10-100 nM to allow the chemical reaction to occur.Subsequently the reacted library is diluted down and the templateoligonucleotide is added in high concentration together with a ligasemixture. Since the library is screened for formation of covalent bonds,the interaction will not be effected by dilution subsequent to thereaction, however the dilution will reduce the random association. Analiquot of the ligation reaction is transferred to a PCR reaction wherethe reverse primer contains a 5′ biotin modification and the forwardprimer a 5′ Cy5 label. The PCR products are then hybridized to a tagmicroarray containing all different tag combinations. The hybridizationto the microarray is designed according to FIG. 6( a).

Ten different molecules of interest are investigated, M₁, M₂, . . . ,M₁₀. These molecules are used to create 20 different interactors dividedinto two pools; one pool with free 3′ ends and one with free 5′ ends.Each molecule of interest is incorporated in two separate interactorsand the identifying sequence elements in these interactors are notidentical. The reaction between two molecules of interest will promoteintra molecular joining of the respective NAMs during the ligation step.These associated oligonucleotides are then detected by microarrayanalysis. The microarray comprise 100 different features, each featurerepresenting a combinations between two different tags. All interactionsexcept the homo interactions are represented by two tag combinations onthe tag microarray. Subsequent to microarray detection the signalintensity of the microarray features is compared within the microarrayand between experiments with different reaction conditions. Threedifferent reaction conditions are investigated, one comprised only ofreaction buffer, one comprised of reaction buffer and inclusion of achemical catalyst, and the last one comprising reaction buffer and aenzyme catalyst, which is presumed to enable reactions between thecurrent substrates.

Results:

Results are illustrated in FIG. 7. The figure schematically illustratesa microarray comprising tags complementary to combinations ofidentification sequence elements. The tags are designated 1 to 10 wheretag 1 corresponds to the interactor comprising molecule of interest M₁and so forth. Thus the interactions between two molecules can bededucted from the signal intensity of the respective tag combination. Inreaction buffer M₆ reacts weakly with molecules of interest M₂ throughto M₆, the last interaction representing an homo interaction. When achemical catalyst is added reactions occur of moderate intensity betweenM₂-M₈, M₆-M₅, and M₃-M₃, the last one also representing a homointeraction. When the chemical catalyst is exchanged to an enzymatic onereaction of high intensity occur, M₉-M₆. The relative reactionefficiencies of the reactions can be interpreted from the arrayintensities, where a high intensity corresponds to an efficientreaction.

Example 4 Enhancement for Microarray Analysis

To facilitate detection of associated oligonucleotides the nucleic acidto be analyzed may be modified to suit the readout platform. Thismodification may include, e.g., modifying enzymes or biochemicalmodification. In the specific case of tag microarray mediated detectionit is desirable to hybridize single stranded DNA. Non limiting examplesof generation of single stranded DNA include: amplifying the associatedoligonucleotides with RCA which produces a single stranded amplificationproduct comprised of concatemeric amplicon repeats; and single strandedDNA generation subsequent to PCR amplification by, e.g., utilization ofone biotinylated primer and streptavidine affinity purification, PCRwith one phosphorylated primer and subsequent lambda exonucleasetreatment or performing PCR with one primer in excess, also known asasymmetric PCR or Linear After The exponential (LATE) PCR.

Depending on the qualitative design of the microarray detection, thesingle stranded DNA may be further modified by, e.g, biochemical orenzymatic modification. One specific example of quantitative design isthe introduction of a ligation step involving two identificationelements on the tag array to form a circular nucleic acid. To enableligation of the two identification elements to the microarray tagoligonucleotide, a nucleic acid has to be generated that comprises oneidentification element in the 3′ end and one in the 5′ end. Downstreamof single stranded DNA generation the single stranded DNA may be cleavedwith a restriction enzyme next to the identification elements.Restriction enzyme digestion of single stranded nucleic acid may beachieved by hybridization of oligonucleotides comprising the restrictionenzyme recognition sequence complementary to the amplification products.In one specific embodiment the enzyme comprises a type IIS restrictionendonuclease and in a further specific embodiment the Type IISrestriction enzyme comprises MlyI.

One general design embodiment of the NAMs provides an associatedoligonucleotide, downstream of association, comprising the associationsequence in the middle flanked by two identification elements which, inturn, are flanked by general sequence in which specific examples can beutilized for amplification by, e.g., PCR or RCA. The associatedoligonucleotide may be enhanced to comprise a single strandedamplification product by, for example, the non limiting methodsdescribed above.

This single stranded amplification product may then comprise theassociation sequence in the middle flanked by two identificationelements which in turn are flanked by a general sequence (FIG. 8). Thegeneral sequence may be designed to comprise the recognition sequencefor a restriction enzyme, more specifically a type IIS restrictionendonuclease, and in a more specific example comprises the type IISrestriction enzyme, MlyI. In one specific embodiment of generating asingle stranded nucleic acid comprising the identification elements inthe 3′ and 5′ ends, the nucleic acid may be designed so the restrictionenzyme cleavage occur right between the general sequence contacted bythe hybridizing oligonucleotide and the single stranded identificationelement. In a very specific embodiment, the restriction enzyme comprisesMlyI. This results in single stranded nucleic acid with a generalsequence in the middle and the identification elements in the 3′ and 5′ends.

This nucleic acid may be hybridized and/or ligated to a tag microarraycomplementary to the identification elements, to form a circular nucleicacid as illustrated in FIG. 5( c). If the nucleic acid is ligated to thetag microarray to form a circular nucleic acid the structure may serveas substrate for RCA amplification. In a specific embodiment, RCA isprimed by the microarray tag oligonucleotide and in a more specificembodiment the polymerase is the phi29 polymerase.

Example 5

This example demonstrates that encoding interactors with unique tags anddetecting the combinations formed therefrom on a combinatorial array mayresolve all interactions within a library. A library of interactors isprovided and association of a defined library subset is detected byreadout of the respective tag combinations. MOIs are thiol groups. Thiolgroups can be induced to dimerize pair-wise and form covalent bonds.This may be compared to a reaction screen where reactive compounds areidentified or it can be viewed as formation of an affinity interactionwith infinite affinity.

Experimental Design

The application of resolving pair wise reacted groups within aninteractor library is exemplified. Formation of a covalent bond isinterrogated within a library. Dimerization of thiol groups serves as anexample. Oligonucleotides with thiol groups will dimerize pairwise ifmixed and subsequently allowed to react. Five oligonucleotides withassociation elements in the 3′ end and 5 oligonucleotides withassociation elements in the 5′ end are employed. Two of the 3′ arms and2 of the 5′ arms are equipped with thiol groups. Subsequent todimerization interacting MOI are identified by association of therespective NAMs by ligation (FIG. 5 a). The ligation joins the two tagmotifs encoding the thiol groups. The ligated product can also serve asamplification template for PCR, enabling selective amplification of theligated NAMs. Subsequent to the PCR single stranded DNA is generated byone biotinylated primer and streptavidine support. The buffer for thesingle stranded DNA is changed by a G50 column. The single stranded DNAis then modified to have the two tag motifs in the ends. This isaccomplished by cleaving off the PCR primer sites next to theidentification tags. (FIG. 8) Cleavage is mediated by annealing twooligonucleotides directing the restriction enzyme cleavage and thenadding the restriction enzyme MlyI. The cleaved product will have theidentifying elements in the 3′ and 5′ end respectively. These are thenadded to a microarray comprising oligonucleotides complementary to thetag combinations. The reporter molecules created by the cleavagehybridize to the array oligonucleotide with the correct tags similar toFIG. 6( c). The hybridized DNA is then ligated and a circular substrateforms which can serve as a template for rolling circle replication anddetected by general detection reagents (FIG. 6( g)).

Experimental Procedure

a. Library Construction via dimerization of thiol modifiedoligonucleotides: all oligonucleotides are obtained from commercialvendors, including oligonucleotides with thiol groups for reactionscreening or amine groups for array manufacturing. Each oligonucleotidecomprises one unique tag element and one of two primer sites. A selectedsubset of the oligonucleotides are also equipped with thiol groupsenabling selective dimerization. The mixture comprising thiol modifiedoligonucleotides is dimerized by addition of 1M DTT, facilitatingreduction of the homodimeric oligonucleotides. Subsequent exclusion ofthe DTT is achieved by a buffer exchange column.b. Association and selection by ligation and PCR

A total volume of 5 μl comprising 100 pM of the thiolated (NAM-Thiol-D5,NAM-Thiol-E5, NAM-Thiol-A3, NAM-Thiol-B3 and non thiolatedoligonucleotides (NAM B5, NAM, C5, NAM F5, NAM C3, Nam D3, NAM E3) ismixed to constitute a final volume of 50 μl comprising 1×PCR buffer(available from Invitrogen, Norway), 5 pmol of each PCR primer(FWDprime, REWprime), 1.5 U Taq platinum (available from Invitrogen,Norway), 3 mM MgCl₂, 80 mM ATP, 100 nM of the oligonucleotide LigA, 200nM dNTP and 1 U T4 DNA ligase. The mixture is incubated at 95 C for 2min and then cycled 30 times between 60 C and 95 C.

c. Analysis of the two identification sequences in the associatedoligonucleotides

Generation of Single Stranded DNA

Five μl of Streptavidine coated Dynabeads M-280 (Invitrogen, Norway) iswashed twice in B&W buffer (1M NaCl, 0.5 mM EDTA, 5 mM Tris-HCl pH 7.5)and subsequently the reaction is added to the beads and allowed to bindfor 10 minutes. The beads are then washed twice in 100 μl of B&W buffer.Subsequent to washing the beads are incubated with 5 μl 0.1M NaOH for 5minutes. The NaOH supernatant is subsequently removed and mixed with 5μl 0.1M HCl and 5 μl 0.1M TRIS-HCl pH8.

Buffer Exchange

The eluted and neutralized DNA is added to a G50 column (available fromGeneral Electric) and purified according to the manufacturersinstructions.

Restriction Enzyme Cleavage

10 μl of the column eluate is mixed with 2 μl of S8 buffer (20 mM TrisAc pH 8, 50 mM KAc, 10 mM (NH₄)₂SO₄, 10 mM MgAc₂, 1 mM DTT) 5 U MlyI(available from New England Biolabs), 10 pmol of the oligonucleotidesREVprime and MlyIRE, 2 μg of BSA and 5.5 μl of ddH₂O. The reaction isfirst incubated for 1 h at 37° C. and then at 75° C. for 15 min.

Microarray Manufacturing

Microarrays are manufactured by a GMS 417 microarray printer. Theoligonucleotides are printed on Codelink™ slides (available from GeneralElectric) in a solution comprising 10 μM oligonucleotide, 150 mM NaCO₃pH9, and 0.05% SDS. After printing, the slides are incubated for atleast 1 h in a humid chamber and then blocked with 30 ml 70% EtOH, 70 ml1×PBS and 0.235 g NaBH₄. Slides are washed and dried by centrifugation.

Ligation to Microarrays

Following incubation the reaction is mixed with 3 μl 10× AmpligaseBuffer (Epicentre), 25 μg BSA, 5 U Ampligase (Epicentre) and 23.5 μlddH₂O. The mixture is added to a microarray slide comprising theoligonucleotides Tag3A5B; Tag3A5C, Tag3A5D, Tag3A5E, Tag3A5F, Tag3B5C,Tag3B5D, Tag3B5E, Tag3B5F, Tag3C5D Tag3C5E, Tag3C5F, Tag3D5E, Tag3D5F,Tag3E5F. The reaction is incubated on the slide in a silicon rubberchamber (Genome Res. 2000 July; 10(7):1031-42.) based on a 96 wellformat at 50° C. over night. The slide is then washed in 0.75×TNT buffer(75 mM Tris-HCl, pH 7.5, 0.1125 M NaCl, and 0.0375% Tween 20) rinsed in0.1×SSC and dried by centrifugation.

Rolling Circle Amplification and Detection on Microarrays

A reaction mixture comprising 1×Phi29 buffer (available from Fermentas),25 μg BSA, 0.2 mM dNTP, 10 U Phi29 in the final volume of 50 μl wasadded to the slide and incubated for 1 h at 37° C. The slide is thenwashed in 0.75×TNT buffer, rinsed in 0.1×SSC and dried bycentrifugation.

The slide is incubated with 50 μl comprising 2×SSC, 0.1×SDS and 10 nM ofthe oligonucleotide DET at room temperature. The slide is then washed in0.75×TNT buffer, rinsed in 0.1×SSC and dried by centrifugation.

Data Acquisition and Analysis

The slide is scanned in a Genepix™ scanner and the data acquired byGenepix pro 5.0 exported and analysed in Microsoft Excel® (MicrosoftOffice 2003). Signal-to-Noise was calculated by dividing the Featuresignal with the average signal of all features.

d. Results

The microarray features are represented in FIG. 9. The grid representsthe combination of the different oligonucleotide tag combinations. Thefour microarray features representing combinations of the interactorsequipped with thiol groups show increased signal to noise, due to theirinteraction. The other tag pairs represented in the library and on thearray display low signal to noise, however significant background isvisible in one feature due to cross hybridization of the detectionoligonucleotide.

1. A method of detecting functional interactions between at least twomolecules of interest, the method comprising: a. forming a plurality ofinteractors by coupling each molecule of interest with at least onenucleic acid moiety, the nucleic acid moiety comprising anidentification sequence element and an association element; b. forming aplurality of cis-reactive cells wherein a cis-reactive cell comprises atleast two interactors bound in proximity to one another by an associatedoligonucleotide formed from the association between at least two nucleicacid moieties, wherein the associated oligonucleotide comprises at leasttwo identification elements derived from the at least two nucleic acidmoieties; c. subjecting the plurality of cis-reactive cells toconditions which stimulate a desired functional interaction having adetectable trace; d. selecting all cis-reactive cells exhibiting thedetectable trace; and e. subjecting the associated oligonucleotides fromthe cis-reactive cells selected in step (d) to an analysis that permitsdetection of the at least two identification sequence elements.
 2. Themethod of detecting functional interactions between at least twomolecules of interest as recited in claim 1, wherein selecting allcis-reactive cells exhibiting the detectable trace is accomplished viaselective separation or selective detection of the cis-reactive cells.3. The method of detecting functional interactions between at least twomolecules of interest as recited in claim 2, wherein selecting allcis-reactive cells exhibiting the detectable trace is accomplished byaffinity purification.
 4. A method of detecting functional interactionsbetween at least two molecules of interest as recited in claim 1,further comprising enhancing the selected associated oligonucleotides byPCR amplification.
 5. A method of detecting functional interactionsbetween at least two molecules of interest as recited in claim 1,wherein coupling is covalent or noncovalent.
 6. A method of detectingfunctional interactions between at least two molecules of interest asrecited in claim 5, wherein coupling is covalent.
 7. A method ofdetecting functional interactions between at least two molecules ofinterest, as recited in claim 1, wherein at least one molecule ofinterest comprises a protein, a protein domain, a peptide, a peptoid, afunctional enzyme, a small molecule, a polymer, a non-proteinaceouscellular product, a virus particle, a cell metabolite, a lipid, acarbohydrate, a nucleic acid, an inorganic compound, a reactivecompound, or any combination thereof, however, wherein not all moleculesof interest are nucleic acids.
 8. A method of detecting functionalinteractions between at least two molecules of interest, as recited inclaim 7, wherein at least one molecule of interest comprises at leastone protein.
 9. The method of detecting functional interactions betweenat least two molecules of interest as recited in claim 8, wherein theprotein is an expression product of a nucleic acid molecule, and furtherwherein at least some of the plurality of interactors are formed bycoupling the protein with a nucleic acid moiety comprising anidentification sequence element derived from the nucleic acid molecule.10. A method of detecting functional interactions between at least twomolecules of interest, as recited in claim 7, wherein the at least twomolecules of interest comprises at least one protein and at least onenucleic acid.
 11. A method of detecting functional interactions betweenat least two molecules of interest, as recited in claim 1, whereindetecting further comprises quantifying the functional interactions. 12.A method of detecting functional interactions between at least twomolecules of interest, as recited in claim 1, wherein the analysiscomprises high throughput screening.
 13. The method of detectingfunctional interactions between at least two molecules of interest asrecited in claim 12, wherein the high throughput screening comprises amicro array analysis.
 14. The method of detecting functionalinteractions between at least two molecules of interest as recited inclaim 1, wherein the analysis comprises nucleic acid sequencing.
 15. Themethod of detecting functional interactions between at least twomolecules of interest as recited in claim 14, wherein the analysiscomprises sequencing by synthesis.
 16. The method of detectingfunctional interactions between at least two molecules of interest asrecited in claim 1, wherein association is via ligation.
 17. The methodof detecting functional interactions between at least two molecules ofinterest as recited in claim 1, wherein association is viapolymerization.
 18. The method of detecting functional interactionsbetween the at least two molecules of interest as recited in claim 1,wherein the conditions for stimulating the functional interactionbetween the at least two molecules of interest comprises manipulatingconditions to maximize cis reactivity and to minimize trans reactivitybetween the at least two molecules of interest.
 19. The method ofdetecting functional interactions between at least two molecules ofinterest as recited in claim 12, wherein the selective detectioncomprises labeling the cis-reactive cells with an affinity reagent. 20.The method of detecting functional interactions between at least twomolecules of interest as recited in claim 2, wherein the selectivedetection comprises introducing a modified molecule wherein themodification permits the selective detection.